Acute inhibition of class I myosins and Myo1c depletion perturbs the expulsion of VWF. (A) Schematic of Myo1c domains and mechanism of inhibition by PCLP. (B) Pharmacological inhibition of the ATP binding domain with 10 to 40 μM PCLP reduces VWF release. (n = 6) ∗P < .05; ∗∗P < .01, ratio paired t test. (C) Schematic of live cell imaging approach to study WPB fusion dynamics and VWF expulsion. Scale bar, 1 μm. (D) HUVECs were electroporated with the P.sel.lum.mCherry and GFP-VWF constructs and imaged by confocal microscopy. Preincubation with 20 μM PCLP increased the time taken for VWF to be expelled after loss of the fusion marker (P.sel.lum.mCherry). Paired t test, ∗∗P < .01. (n = 3; DMSO: 9 cells, 63 events; PCLP: 9 cells, 38 events; mean ± standard error of the mean [SEM]). (E) A frequency distribution of events. (F) LUC and Myo1c KD HUVEC were used to test whether these effects were specific to Myo1c or a broader effect of class I myosins. Western blotting determined the efficiency of target protein KD. (G) Myo1c siRNA depletion increased the time taken for VWF to be expelled after loss of P.sel.lum.mCherry. ∗P < .05; paired t test.